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Genes to Cells (2006) 11, 269-279. doi:10.1111/j.1365-2443.2006.00933.x
© 2006 Blackwell Publishing or its licensors
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Activation of p38 mitogen-activated protein kinase and partial reactivation of the cell cycle by cis-4-methylsphingosine direct postmitotic neurons towards apoptosis

Sven Naetzker, Nadine Hagen and Gerhild van Echten-Deckert*

Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
As shown before in three different cell types, cis-4-methylsphingosine is a synthetic, membrane permeable, pro-drug, that is taken up by cells and phosphorylated to a metabolically stable cis-4-methylsphingosine-phosphate. The synthetic compound mimicked the mitogenic effect of sphingosine-1-phosphate (S1P) in Swiss 3T3 fibroblasts, but induced apoptosis in B104 neuroblastoma cells. We now investigated its effect in differentiated primary cultured neurons. In contrast to S1P, which had no effect on growth of these postmitotic cells, cis-4-methylsphingosine-phosphate induced apoptosis. Interestingly, both compounds stimulated extracellular regulated kinase (ERK) and also p38 mitogen-activated protein kinase (MAPK). Additionally, both compounds induced an increased expression of cyclin D1 but not of cyclin E. Our results document that the different physiological effects, apoptosis in the case of the accumulating metabolically stable synthetic compound vs. no apoptosis in the case of the short-living S1P, rely only on nuances of impact. In other words both sphingoid phosphates affect similar pathways albeit in a sustained and more pronounced manner in case of the metabolically stable synthetic compound. Experiments with several pharmacological inhibitors indicate that cis-4-methylsphingosine-phosphate-induced neuronal apoptosis is mediated on the one hand by a caspase dependent and p38 MAPK forwarded pathway and on the other hand by an abortive reactivation of the cell cycle, a caspase independent process.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Sphingosine-1-phosphate (S1P), the major mammalian sphingoid long-chain base phosphate has been shown to regulate a wide range of physiological processes (Pyne & Pyne 2000; Spiegel & Milstien 2003). Since S1P-mediated pathways are primarily proliferative and anti-apoptotic, this molecule evolved to a generally accepted survival signal (Spiegel & Milstien 2003). Nevertheless, elevation of endogenous sphingoid long-chain base phosphates was reported to kill yeast cells (Zhang et al. 2001). Also mammalian cells including HepG2 (Hung & Chuang 1996), hepatic myofibroblasts (Davaille et al. 2002) and hippocampal neurons (Moore et al. 1999) were shown to undergo apoptosis following S1P treatment. Furthermore, in myoblasts the anti-mitogenic effect of S1P was paralleled by stimulation of myogenic differentiation (Donati et al. 2005). Interestingly, S1P can be rapidly dephosphorylated to cytotoxic sphingosine (Ohta et al. 1994; Linn et al. 2001), which in turn can be acylated to ceramide, a well-known anti-proliferative and pro-apoptotic molecule (Hannun & Luberto 2000). The closely interconnected metabolism of these antagonistically bioactive sphingolipids might hamper the study of their signaling function. One approach to overcome this difficulty is to manipulate expression of genes encoding enzymes involved in S1P and ceramide metabolism, an approach applicable especially in yeast cells (Mao et al. 1999; Birchwood et al. 2001; Zhang et al. 2001), invertebrate cells including Drosophila (Herr et al. 2004), and Caenorhabditis elegans (Mendel et al. 2003) and mammalian cell lines (Kihara et al. 2003; Reiss et al. 2004). In primary cultured cells that are highly differentiated and postmitotic an alternative strategy, namely the employment of metabolically stable synthetic analogs of the signaling molecules, is recommended. cis-4-Methylsphingosine, a synthetic sphingosine analog was shown to be taken up by cells when added to the culture medium but unlike the physiological archetype it is not N-acylated to the respective ceramide but exclusively phosphorylated to the corresponding cis-4-methylsphingosine-phosphate (van Echten-Deckert et al. 1997). In contrast to S1P, cis-4-methylsphingosine-phosphate is metabolically stable thus accumulating intracellularly (van Echten-Deckert et al. 1997, 1998; Nätzker et al. 2002). In quiescent Swiss 3T3 fibroblasts cis-4-methylsphingosine-phosphate mimicked the proliferative effect of its short-living physiological counterpart S1P (van Echten-Deckert et al. 1998). However, unlike S1P it induced apoptosis in rapidly growing B104 neuroblastoma cells (Nätzker et al. 2002). The cell type specific effect on survival was accompanied by a cell type specific effect on MAPK regulation. Thus in Swiss 3T3 fibroblasts both sphingoid phosphates stimulated ERK (Wu et al. 1995; Rani et al. 1997; Nätzker et al. 2002). In B104 cells also both sphingoid phosphates stimulated p38 MAPK but only the synthetic long living compound simultaneously inhibited ERK in addition (Nätzker et al. 2002). Using different MAPK inhibitors we have demonstrated that exactly the latter condition, namely simultaneous and opposite regulation of ERK and of p38 MAPK was essential for induction of apoptosis in B104 cells.

S1P was shown to act both inside and outside of cells (Spiegel & Milstien 2003). Its role in cellular growth appears, however, to be independent of S1P receptors (Van Brocklyn et al. 1998; Spiegel & Milstien 2003). Hence, cis-4-methylsphingosine, a synthetic pro-drug that only after intracellular phosphorylation yields a metabolically stable S1P analog was used to study S1P mediated signaling pathways in postmitotic neurons. We report here that although both sphingoid phosphates affect similar MAPK pathways and similar cell cycle regulators only the synthetic compound induces neuronal apoptotic cell death thus indicating the importance of impact of drug effects on molecular level for physiological consequences.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Phosphorylated cis-4-methylsphingosine induces caspase dependent apoptosis in primary cultured cerebellar neurons

The viability of neurons was assessed after 24 h incubation in the presence of 10 µM of the synthetic cis-4-methylsphingosine or the natural sphingolipids sphingosine and S1P, respectively. As illustrated in Fig. 1A, a marked decrease in cell viability (by about 40%) was observed only in the presence of the synthetic compound. We have shown before that in contrast to sphingosine, which is primarily used as a biosynthetic precursor for ceramide and complex sphingolipids including gangliosides that are highly abundant in these cells, cis-4-methylsphingosine is exclusively phosphorylated to the respective sphingoid phosphate (van Echten-Deckert et al. 1997). In contrast to S1P, the synthetic sphingoid phosphate with a cis instead of the trans configuration of the 4,5 double bond and an additional methyl group at carbon atom 4 is a poor substrate for S1P lyase thus accumulating intracellularly. As illustrated in Fig. 1B, phosphorylated cis-4-methylsphingosine was detectable in abundance in primary cultured neurons. Relative to the amount of endogenously labeled S1P of control cells, the amount of phosphorylated cis-4-methylsphingosine was found to be elevated about 200-fold after 1 h of incubation, rising to about 650-fold after 24 h of incubation. To find out whether reduction of cell viability by cis-4-methylsphingosine was due to apoptotic cell death, its effect on integrity of genomic DNA was studied. As shown in Fig. 1Ccis-4-methylsphingosine clearly induced oligonucleosomal fragmentation of DNA, which represents a generally accepted hallmark of apoptosis. Furthermore, only in the presence of the synthetic compound a marked activation of effector caspases, known to mediate apoptotic cell death was observed (Fig. 1D). However, no changes, neither of caspase activity nor of DNA integrity could be observed in cells treated with sphingosine or S1P when compared with untreated controls (Fig. 1C,D). Thus, cis-4-methylsphingosine-phosphate, in contrast to S1P induces caspase-dependent apoptotic cell death in primary cultured cerebellar neurons. As illustrated in Fig. 2 there is a good correlation between the time courses of cis-4-methylsphingosine induced caspase activation, DNA fragmentation and loss of cell viability.


Figure 1
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  		Figure 1  Induction of apoptosis in primary cultured neurons by accumulating cis-4-methylsphingosine-phosphate. Cells were treated with vehicle (controls) or 10 µM of the indicated sphingoid. After 24 h (A) cell viability and (D) caspase activity were determined or (C) genomic DNA was analyzed as described in Experimental procedures. (B) Structure and phosphorylation of cis-4-methylsphingosine. Sphingoid phosphates in cells treated for the indicated times with vehicle (controls) or cis-4-methylsphingosine (10 µM) were determined in phosphate free DMEM supplemented with 32Pi (10 µCi/mL) as described in Experimental procedures. Abbreviations are as follows: C, controls; cis, cis-4-methylsphingosine; cis32P, cis-4-methylsphingosine-phosphate; M, marker DNA; So, sphingosine; S1P, sphingosine-1-phosphate. Lyase cleavage site is shown with a dashed arrow.

 

Figure 2
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  		Figure 2  Time course of apoptotic events after treatment of primary cultured neurons with cis-4-methylsphingosine (10 µM). After the indicated times cell viability and caspase activity were determined or genomic DNA was analyzed as described in Experimental procedures. *significantly different from control value (P < 0.05).

 
Phosphorylated cis-4-methylsphingosine enhances activation of ERK and p38 MAPKs. As shown before in B104 neuroblastoma cells cis-4-methylsphingosine-phosphate-induced apoptosis required an opposite regulation of ERK and p38 MAPKs (Nätzker et al. 2002). We have therefore investigated the effect of cis-4-methylsphingosine on the regulation of these two MAPKs in postmitotic cerebellar neurons. The phosphorylation (activation) state of both ERK and p38 MAPK was assessed over an extended time period (15 min to 24 h) following treatment of neurons with 10 µM of cis-4-methylsphingosine and S1P, respectively. As illustrated in Fig. 3Acis-4-methylsphingosine induced a significant activation of ERK that persisted at about 150% up to 6 h, gradually returning to basal levels after 12-18 h. Interestingly, S1P also induced an increase in ERK MAPK activation, which in contrast to the sustained ERK activation following cis-4-methylsphingosine treatment peaked faster but transient, returning to basal levels already after 1 h (Fig. 3A).


Figure 3
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  		Figure 3  Time course of MAPK phosphorylation in the presence of sphingoid phosphates. Cells were incubated in the absence (controls) or presence of 10 µM of S1P or cis-4-methylsphingosine (cis) for the indicated times. (A) Phosphorylated ERK (pERK) or (B) phosphorylated p38 MAPK (pp38) were determined by immunoblotting with a specific antibody, respectively. Blots were stripped and re-probed with an antibody specific for total ERK (ERK) or total p38 MAPK (p38), respectively, to confirm equal loading. Western blots were densitometrically evaluated as described in Experimental procedures. Data are shown relative to the mean value of time 0 (100%). *significantly different from the control value (P < 0.05).

 
The effect of cis-4-methylsphingosine on p38 MAPK activation was similar to that found for ERK. As becomes clear from Fig. 3B the marked activation of p38 MAPK by the synthetic compound persisted for up to 6 h, whereas activation of this MAPK by S1P reached an early maximum of about 150% relative after 15 min and then immediately returned to basal levels.

p38 MAPK mediates caspase promoted apoptotic cell death in cis-4-methylsphingosine treated postmitotic neurons

Several studies concerning neuronal cell death report on the involvement of the p38 MAPK pathway in neuronal apoptosis (for review see Takeda & Ichijo 2002). To clarify the relevance of this pathway in cis-4-methylsphingosine induced neuronal apoptosis, we used SB239063, a highly potent pharmacological inhibitor shown to specifically suppress p38 MAPK (Barone et al. 2001). As illustrated in Fig. 4A, SB 239063 alone had no effect, neither on cell viability nor on integrity of genomic DNA, nor on the activity of effector caspases when compared with untreated control cells. However, simultaneous addition of the inhibitor and of cis-4-methylsphingosine significantly reduced the effects of the latter on both cell viability and DNA cleavage, whereas caspase activity was completely abrogated (Fig. 4A). To verify persistence of apoptosis despite an abolished caspase activity, we determined apoptosis in the presence of the pan-caspase inhibitor Z-VAD-fmk. The results are shown in Fig. 4B and correspond to those obtained with the p38 MAPK inhibitor SB239063. Notably, inhibition of ERK with PD98059 (10–100 µM) had no effect on cis-4-methylsphingosine induced neuronal apoptosis (not shown).


Figure 4
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  		Figure 4  Inhibition of p38 MAPK and of caspase activity partially reduces cis-4-methylsphingosine-induced apoptosis in primary cultured neurons. Cells were cultured in the presence (C) of vehicle (controls), of (A) 5 µM SB239063 (SB) or of (B) 100 µM of Z-VAD-fmk (ZVAD), or of 10 µMcis-4-methylsphingosine (cis) or of both additions as indicated. After 24 h cell viability and caspase activity were determined or genomic DNA was analyzed as described in Experimental procedures. *significantly different from cells incubated with cis-4-methylsphingosine alone and also from the controls (P < 0.05).

 
Reactivation of the cell cycle machinery promotes cis-4-methylsphingosine-phosphate-induced apoptosis in postmitotic neurons

About 10 years ago it was proposed that pro-apoptotic agents might induce a dedifferentiation process in mature neurons including reactivation of quiescent cell cycle functions (Heintz 1993). According to this ‘cell cycle theory of neuronal apoptosis’ reactivation of neuronal cell cycle remains incomplete, thus resulting in conflicting intracellular signaling that ultimately drives the neuron into cell death. To test whether an inappropriate activation of the cell cycle is involved in cis-4-methylsphingosine-phosphate-induced neuronal apoptosis, we studied its effect on the expression of cyclin D1. Cyclin D1 and its associated kinase activity CDK4 (cyclin dependent kinase 4) are major regulators of early steps in cell cycle progression, e.g. re-entry of quiescent cells into the cell cycle and progression through G1 phase (Baldin et al. 1993). Notably, the involvement of cyclin D1 in apoptosis of cerebellar granule cells as well as in other postmitotic neurons has been shown before (Freeman et al. 1994; Sakai et al. 1999). As shown in Fig. 5, a sustained and significant up-regulation of cyclin D1 expression in cerebellar neurons incubated with 10 µM of cis-4-methylsphingosine was observed. This elevated expression persisted up to 12 h, returning to control levels after 24 h (not shown). By contrast, the short-living physiological counterpart S1P induced only a transient increase of cyclin D1 expression that peaked after 6 h (Fig. 5). Surprisingly, the expression of cyclin E, a major target of cyclin D1 was not affected by either of the compounds (not shown). These results suggest an abortive reactivation of the cell cycle machinery in the presence of sphingoid phosphates. To evaluate the possibility of an involvement of cyclin D1 elevation in cis-4-methylsphingosine-phosphate-induced neuronal apoptosis, cell cycle blocking cytostatic drugs including 2-bromo-12,13-dihydro-5H-indolo[2,3-1]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (CDK4 inhibitor), a highly specific inhibitor of the cyclin D1/CDK4 complex (Zhu et al. 2003) as well as roscovitine (50 µM) and olomoucine (200 µM) that non-selectively inhibit cyclin/CDK complexes were employed and gave similar results. As illustrated in Fig. 6, the effect of cis-4-methylsphingosine on cell viability and DNA fragmentation was reversed by about 50% in the presence of the specific CDK4 inhibitor (100 nM), even though caspase activation remained unaffected. Importantly, the p38 MAPK inhibitor SB239063 did not affect cis-4-methylsphingosine induced prolonged accumulation of cyclin D1 (Fig. 5), and CDK4 inhibitor did not affect p38 activation by cis-4-methylsphingosine (Fig. 6). Together these results suggest that a p38 and caspase independent reactivation of the cell cycle machinery is involved in cis-4-methylsphingosine-phosphate-induced neuronal apoptotic cell death.


Figure 5
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  		Figure 5  Effect of sphingoid phosphates and of p38 MAPK on cyclin D1 expression. Cells were incubated in the presence of vehicle (control) or 10 µM of S1P or 10 µM of cis-4-methylsphingosine (cis) or 5 µM of SB239063 (SB), or a combination of cis-4-methylsphingosine and SB239063 for the indicated times. Cyclin D1 expression was determined by immunoblotting with a specific antibody. Blots were stripped and re-probed with an anti-{alpha}-tubulin antibody to confirm equal loading. Western blots were densitometrically evaluated as described in Experimental procedures. Data are shown relative to the mean value of time 0 (100%). *significantly different from the control value (P < 0.05).

 

Figure 6
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  		Figure 6  Selective inhibition of cyclin D1/CDK4 complexes reduces cis-4-methylsphingosine-induced apoptosis in primary cultured neurons. Cells were cultured in the presence of vehicle (C), or 100 nM of CDK4 inhibitor (In), or 10 µM of cis-4-methylsphingosine (cis) or both additions as indicated. After 24 h cell viability and caspase activity were determined or genomic DNA was analyzed as described in Experimental procedures. Alternatively, after 4 h phosphorylated p38 MAPK (pp38) was determined by immunoblotting with a specific antibody. Blots were stripped and re-probed with an antibody specific for total p38 MAPK (p38) to confirm equal loading. *significantly different from cells incubated with cis-4-methylsphingosine alone and also from the control value (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
cis-4-Methylsphingosine-phosphate was shown to accumulate in three different cell types including primary cultured cerebellar neurons (van Echten-Deckert et al. 1997), quiescent Swiss 3T3 fibroblasts (van Echten-Deckert et al. 1998) and neuroblastoma B104 cells (Nätzker et al. 2002) upon incubation with the respective free base. Thus, accumulation of this compound in all cell types examined so far suggests that its metabolic stability is primarily related to its molecular structure rather than being cell type specific. Cell type specificity, however, was observed in the effect on cell growth associated with the accumulation of cis-4-methylsphingosine-phosphate. It was previously shown that this compound induced proliferation in quiescent Swiss 3T3 fibroblasts thus mimicking the mitogenic effect of its physiological counterpart S1P (van Echten-Deckert et al. 1998). However, unlike S1P, cis-4-methylsphingosine-phosphate induced a marked apoptotic response in neuroblastoma B104 cells (Nätzker et al. 2002) and as shown in the present study, also in primary cultured cerebellar neurons. In all three cell types the effect of the synthetic as well as that of the natural sphingoid phosphate on cell growth was found to be closely and specifically related with MAPK signaling pathways (Wu et al. 1995; Nätzker et al. 2002). In postmitotic neurons, like in quiescent Swiss 3T3 fibroblasts both sphingoid phosphates affect similar MAPK pathways albeit in a sustained and more pronounced manner in the case of the metabolically stable synthetic compound. As a consequence, in fibroblasts the mitogenic effect of the synthetic compound was three-fold stronger than that of S1P (van Echten-Deckert et al. 1998). Surprisingly, in postmitotic neurons the synthetic compound induced apoptosis whereas S1P had no effect on cell growth. It thus appears that nuances of impact are responsible for the remarkable difference concerning the physiological effect of the two related compounds in postmitotic neurons. Yet more remarkable is the fact that in postmitotic neurons, cis-4-methylsphingosine-phosphate-induced apoptotic cell death could be correlated with the activation of p38 MAPK despite a simultaneous activation of ERK. In neuroblastoma cells, the apoptotic effect of cis-4-methylsphingosine-phosphate was also mediated by activation of the p38 MAPK pathway but a concurrent down-regulation of ERK was crucial for the apoptotic response whereas a simultaneous activation of ERK, as observed in the presence of S1P prevented apoptotic cell death (Nätzker et al. 2002).

p38 MAPK is well known to mediate apoptotic cell death in primary cultured neurons in response to glutamate, potassium withdrawal, and Fas receptor activation (Kawasaki et al. 1997; Yamagishi et al. 2001; Hou et al. 2002). Accordingly, our results indicate that p38 mediates caspase dependent apoptosis in neurons accumulating cis-4-methylsphingosine-phosphate. Based on the time courses of p38 and caspase activation, respectively, and of DNA-fragmentation, a hallmark of apoptotic cell death induced by the synthetic analog, it appears that p38 activation is an early event with an apparently priming function as observed before in this cell type (McLaughlin et al. 2001). Although MAPK phosphorylation returns to basal levels after 6 h, apoptotic signaling once started is obviously irreversible. The finding that cis-4-methylsphingosine-induced apoptosis cannot be completely abolished by inhibition of p38 MAPK or of caspases, respectively, points to a collateral, caspase-independent apoptotic cell death in primary cultured neurons. Simultaneous induction of both, capase-dependent and -independent apoptotic cell death by one particular stimulus has already been reported for neuronal cells (Stefanis et al. 1999; Volbracht et al. 2001; Zhan et al. 2001). A caspase independent process involved in neuronal apoptosis was found to be an abortive cell cycle re-entry triggered by pro-apoptotic stimuli in postmitotic neurons (Padmanabhan et al. 1999; Sakai et al. 1999; Martin-Romero et al. 2000). Re-entry of postmitotic, terminally differentiated or of quiescent cells into the G1 phase is promoted by CDK4, which is active only when associated with cyclin D1 (Baldin et al. 1993). The latter was clearly up-regulated by both sphingoids, albeit in a more sustained fashion by the metabolically stable synthetic compound (12 h vs. 6 h). From studies with a selective inhibitor of cyclin D1/CDK4 complex and two non-selective CDK inhibitors it became clear that cell cycle re-entry is correlated with cis-4-methylsphingosine-phosphate-induced neuronal apoptotic cell death. Analysis of the time course of cyclin D1 activation in the presence of S1P and the synthetic analog, respectively, suggests that a sustained activation (12 h) is required to trigger an apoptotic response. These results confirm earlier reports on the role of the cell cycle regulator cyclin D1 in mediating apoptosis in terminally differentiated neurons (Freeman et al. 1994; Kranenburg et al. 1996; Sakai et al. 1999; Martin-Romero et al. 2000). Notably, over-expression of cyclin D1 was sufficient to induce apoptosis in neuroblastoma cells (Kranenburg et al. 1996) but also in non-neural cell types including fibroblasts (Sofer-Levi & Resnitzky 1996). Moreover, it has been shown repeatedly that CDKs and especially CDK4, known to be activated by increased levels of cyclin D1 are essential for apoptotic cell death of cerebellar neurons (Martin-Romero et al. 2000; Verdaguer et al. 2003; Otsuka et al. 2004). In contrast to these studies in which pro-apoptotic agents including potassium withdrawal, doxorubicin and kainic acid were used to induce apoptosis, S1P and cis-4-methylsphingosine-phosphate were shown to induce proliferation in quiescent Swiss 3T3 fibroblasts (Wu et al. 1995; van Echten-Deckert et al. 1998). This proliferative effect was correlated with stimulation of ERK (Wu et al. 1995; Nätzker et al. 2002). Although no correlation of ERK activation and sphingoid phosphate induced neuronal apoptosis could be established in this study it is not clear at the present time whether, apart from stimulation of ERK, conflicting growth signals mediate the failed attempt of cerebellar neurons to re-enter cell cycle. Since unregulated proliferation might be especially dangerous when affecting the central nervous system, one can imagine that in terminally differentiated postmitotic neurons protective strategies have evolved, to prevent or at least hamper a complete reactivation of the cell cycle machinery as triggered by proliferative stimuli. It is known that over-expression of oncogenes frequently triggers apoptosis instead of proliferation in different cell types (Evan & Littlewood 1998) indicating that apoptosis in differentiated cells might involve mechanisms similar to those that promote proliferation in transformed cells. Thus, cell proliferation and cell death, despite representing two diametrically opposed cellular fates, are closely linked and interdependent processes (Lowe et al. 2004). It appears therefore likely that as depicted in Fig. 7 cis-4-methylsphingosine-phosphate-induced neuronal apoptosis is a complex process involving on the one hand a p38 MAPK mediated caspase-dependent pathway and on the other hand results from a failed attempt of postmitotic neurons to re-enter cell cycle. In addition, our study suggests that S1P, a generally accepted pro-survival molecule, in excess might be fatal for postmitotic neurons.


Figure 7
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  		Figure 7  Proposed model for cis-4-methylsphingosine-induced apoptosis in primary cultured cerebellar neurons involving both stimulation of p38 MAPK and reactivation of cell cycle.

 

   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

Six-day-old NMRI (Navy Marine Research Institute) mice were bred in the animal house of the University in Bonn (Germany). cis-4-Methylsphingosine was synthesized by Dr Santiago Figueroa-Perez in the laboratory of Prof R. R. Schmidt, University of Konstanz (Germany) as previously described (Bär et al. 1993). S1P and the ERK inhibitor, PD98059 were purchased from Sigma (Taufkirchen, Germany). Dulbecco's modified Eagle's medium (DMEM), Minimal Essential Medium, trypsin, deoxyribonuclease, bovine serum albumin, and horse serum were obtained from Gibco-BRL (Karlsruhe, Germany), cytosine arabinoside was from Sigma (Taufkirchen, Germany). Antibodies recognizing phosphorylated ERK, total ERK, phosphorylated p38 MAPK and total p38 MAPK were obtained from New England Biolabs (Frankfurt, Germany), while antibodies against cyclin D1 and {alpha}-tubulin were from Santa Cruz Biotechnology (Heidelberg, Germany). Complete Mini Protease Inhibitor was purchased from Roche (Mannheim, Germany). The ApoAlert Caspase-3 Colorimetric Assay Kit was obtained from BD Pharmingen (Heidelberg, Germany) and the CellTiter-Blue Cell Viability Assay from Promega (Mannheim, Germany). SB239063 was kindly provided by GlaxoSmithKline. The CDK4 inhibitor, 2-Bromo-12,13-dihydro-5H-indolo[2,3-1]pyrrolo[3,4-c]carbazole-5,7(6H)-dione was from Calbiochem (Schwalbach, Germany). All other chemicals and supplies were purchased as previously described (van Echten-Deckert et al. 1997).

Cell culture

Granule cells were cultured from cerebella of 6-day-old mice as described before (van Echten-Deckert et al. 1997). Briefly, cells were isolated by mild trypsinization (0.05%, w/v) and dissociated by repeated passage through a constricted Pasteur pipette in a DNase solution (0.1%, w/v). The cells were then suspended in DMEM (containing 10% heat-inactivated horse serum and plated on to poly L-lysine-coated 8 cm2 Petri dishes (6 x 106 cells/dish). Twenty-four hours after plating, cytosine arabinoside was added to the medium (4 x 10–5 M) to arrest the division of non-neuronal cells. After 5–6 days in culture, cells were used for metabolic studies. Sphingolipids were provided as complexes with fatty acid free bovine serum albumin.

Cell viability assay

For quantification of cell death in cultured cells, we employed the CellTiter-Blue Cell Viability Assay that is based on the conversion of resazurin to the fluorescent product resorufin exclusively by metabolically active (viable) cells. Granule cells cultured in 35-mm diameter dishes were treated with 1 mL of the indicated media. After 24 h, 100 µL of CellTiter-Blue reagent (resazurin) were added to each cell culture dish, and incubation continued for 1 h. Then an aliquot of 100 µL from each culture dish was transferred to a 96-well microtiter plate and fluorescence of resorufin was recorded (544Ex/590Em nm). After the viability assay, cells were further processed for genomic DNA analysis.

Genomic DNA analysis

Cell dishes used for cell viability assays were further processed for genomic DNA isolation as previously described (Nätzker et al. 2002). Cells were detached with a cell lifter directly in the respective incubation medium and centrifuged (2000 x g, 10 min, 4 °C), thus collecting both adherent and detached cells. Cell pellets were then resuspended in 200 µL phosphate buffered NaCl solution (PBS; 3 mM KCl, 1.5 mM KH2PO4, 140 mM NaCl, 16 mM Na2HPO4, pH 7.4) and centrifuged (2000 x g, 10 min, 4 °C) again. Genomic DNA was purified using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the protocol of the provider. Finally, 2 µg of isolated DNA per sample were loaded on to a 1.4% agarose gel prepared from Agarose SMG (AppliChem, Darmstadt, Germany). After electrophoresis, DNA was visualized under UV light using ethidium bromide.

Effector caspase assay

To collect both adherent and detached cells, neurons were harvested directly in the respective medium and centrifuged (2000 g, 10 min, 4 °C). The cell pellet was washed by resuspension in 200 µL PBS and centrifugation (2000 g, 10 min, 4 °C). Effector caspase assay was performed according to the provider's protocol. Cell pellets were resuspended in 50 µL of cold lysis buffer and incubated on ice for 15 min. After addition of 50 µL of reaction buffer containing dithiotreitol (10 mM) and DEVD-p-nitroanilin substrate (50 µM) samples were incubated for 1 h at 37 °C in the absence of light. Absorbance was measured at 405 nm. Effector caspase activities are expressed relative to untreated controls.

SDS-PAGE and Western blot analysis of MAPKs

Cells were harvested in ice-cold PBS, centrifuged (2000 g, 10 min, 4 °C) and incubated with lysis buffer containing 20 mM HEPES, pH 7.4, 100 mM sodium chloride, 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 50 mMß-glycerophosphate, 2 mM sodium orthovanadate, 2 mM EDTA, and Complete Mini Protease Inhibitor (1 tablet for 10 mL buffer) followed by sonication for 15 s. Cell debris was removed by centrifugation for 15 min at 18 000 g and 4 °C. Twenty micrograms of total cellular protein were separated on 10% SDS-polyacrylamide gels and then blotted to enhanced nitrocellulose membranes. Membranes were blocked with a buffer containing non-fat dry milk (5% w/v) and Tween-20 (0.05% v/v) for 1 h at room temperature followed by incubation with the respective primary antibody diluted in Tris-buffered NaCl solution (TBS, 137 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 0.1% Tween-20 overnight at 4 °C. Antibodies specific for phosphorylated or total ERK and p38 MAPK were diluted 1 : 10 000, whereas the antibody against cyclin D1 was diluted 1 : 200. The membranes were rinsed with TBS, 0.1% Tween-20, and incubated for 2 h at room temperature in horseradish peroxidase-conjugated goat anti-rabbit antibody diluted 1: 20 000 in TBS with 0.1% Tween-20 except membranes treated with the antibody specific for cyclin D1. The latter antibody was provided already conjugated to horseradish peroxidase. Bound antibodies were visualized using ECL on X-ray films.

To confirm equal amounts of protein loaded for each sample, blots were subsequently stripped using a low pH-buffer containing 0.2 M glycine, pH 2.2, 0.1% SDS, and 1% Tween-20 and reprobed with an antibody specifically recognizing total (phosphorylated and unphosphorylated) MAPK or {alpha}-tubulin for membranes first treated with anti-cyclin D1 antibody. Western blots were densitometrically evaluated using the AlphaDigiDoc gel documentation system (Biorad, München, Germany). In each experiment the ratio of absorbance of phosphorylated vs. total MAPK or cyclin D1 vs. {alpha}-tubulin was calculated. The relative activation (phosphorylation) of MAPK or expression of cyclin D1 obtained after treatment of cells was then expressed relative to mean values obtained at time point 0 (100%).

32Pi labeling and extraction of phosphorylated sphingoid bases

Primary cultured neurons were washed with phosphate-free DMEM and then incubated in the same basic medium supplemented with 10 µCi/mL 32Pi for 24 h. The cells were then treated with 10 µM of cis-4-methylsphingosine or vehicle for the indicated times. Sphingosine phosphates were extracted as described in detail previously (van Echten-Deckert et al. 1997). Phosphorylated sphingosines were resolved by thin layer chromatography in 1-butanol/methanol/acetic acid/water (80 : 20 : 10 : 20, by volume), visualized by autoradiography, identified by their RF value and quantitatively evaluated using the bio-imaging analyzer Fujix Bas 1000 using software TINA 2.09 (Raytest, Straubenhardt, Germany).

Protein determination

Cell protein was quantified as previously described using bovine serum albumin as standard (Nätzker et al. 2002).

Presentation of data and statistics

All experiments were repeated at least three times with consistent results. Data are expressed as means ± SD, and normalized to time 0 (set as 100%). Statistical analysis was performed using Student's t-test. Results presented as DNA or protein gels or as thin layer chromatography image, correspond to data obtained with at least three different cell preparations.


   Acknowledgements
 
We thank Prof K. Sandhoff for continual encouragement and Andrea Raths for excellent technical assistance. We especially acknowledge Dr Santiago Figueroa-Perez and Prof RR Schmidt for providing cis-4-methylsphingosine and Dr Frank C. Barone from GlaxoSmithKline for providing SB 239063. This study was supported by the Deutsche Forschungsgemeinschaft (Grant EC 118/2-1).


   Footnotes
 
Communicated by: Fumio Hanaoka

* Correspondence: E-mail: g.echten.deckert{at}uni-bonn.de


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 22 June 2005
Accepted: 20 November 2005




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